This invention relates to a process for measuring the amount of endotoxin or (1.fwdarw.3)-.beta.-D-glucan (referred to hereinafter as .beta.-glucan) contained in a sample which comprises reacting the endotoxin or .beta.-glucan with amebocyte lysate of horseshoe crabs and analyzing the reaction mixture by a kinetic turbidimetric method.
Endotoxin is a substance present in cell walls of Gram-negative bacteria and has various biological activities such as fervescence and the like. Therefore, when medical devices which contact directly with drugs or blood are contaminated with endotoxin, a grave result is brought about in some cases even if the amount of the endotoxin is very slight. Hence, the contamination with endotoxin must be controlled strictly. Also, for early diagnosis of septicemia due to Gram-negative bacteria, the quantitation of the endotoxin in blood has been tried.
Also, the .beta.-glucan is a substance which is known to exist as a skeleton constituent of cell walls of yeasts and molds, as an important polysaccharide component of many basidiomycetes fruit bodies (mushroom), as an eluate component from a membrane or the like used in the hemodialysis, or the like. The biological activity of the .beta.-glucan is not so clear as in the case of the endotoxin; however, it has been considered that early diagnosis of mycosis and detection of contamination of medical devices with fungi be made possible by measuring the amount of the .beta.-glucan.
Each of the endotoxin and .beta.-glucan is known to react with a solution containing amebocyte lysate of horseshoe crabs (referred to hereinafter as the AL solution) to activate an enzyme and cause gelation reaction. Utilizing such properties, various methods for measuring the amount of the endotoxin or .beta.-glucan contained in a sample have been developed and utilized.
Representative examples of the measurement method utilizing the gelation reaction which is caused between the AL solution and the endotoxin (or the .beta.-glucan) include a so-called gel-clot method in which it is visually confirmed whether or not gelation is caused and so-called kinetic turbidimetric method such as a method for determining the amount of the endotoxin (or .beta.-glucan) contained in a sample on the basis of the relationship between the endotoxin (or .beta.-glucan) concentration and the gelation time determined by measuring the time required until a varied degree of the transmitted light amount ratio (R.sub.t) [the ratio between the transmitted light amount at the initial stage (I.sub.0) and the transmitted light amount (I.sub.t) after the time t elapsed from the mixing of the sample with the AL solution or from the lapse of predetermined time from the mixing of the sample with the AL solution, R.sub.t =I.sub.t /I.sub.0 ] which ratio is reduced by gelation reaction or a varied degree of a logarithmic value of the ratio R.sub.t reaches a predetermined value, a method for determining the amount of the endotoxin (or .beta.-glucan) in a sample from the relationship between the endotoxin (.beta.-glucan) concentration and the onset time determined by measuring the time required until a varied degree of the change of transmittance or absorbance due to gelation reaction reaches a predetermined value, etc.
Among these methods, the kinetic turbidimetric method is simpler and has a higher sensitivity than the gel-clot method and hence has been widely used.
However, the detection limit of endotoxin by the current kinetic turbidimetric method is usually about 0.001EU/ml, and hence, a measuring method having such a high sensitivity that much less endotoxin can be detected has been desired.
On the other hand, U.S. Pat. No. 4,221,865 disclose a process for determining endotoxin improved in the measurement sensitivity and enhance the reproducibility in a nephelometric method by allowing both ionic surfactant and suspending agent or a polymer having simultaneously the properties of the two to be present during the reaction between the AL solution and the endotoxin (or .beta.-glucan). According to this U.S. Patent, an ionic surfactant is added so as to stop the coagulation of Limulus protein, and a suspending agent is added to stabilize pharmaceutical or food emulsions and colloidal solutions.
However, this method must use two reagents of surfactant and suspending agent, so that there are such problems that the operation becomes complicated and the possibility that the AL solution is contaminated with the endotoxin or .beta.-glucan present in the reagents is high. Therefore, a further improvement is desired even at present.